Phosphodiesterase type 3 (PDE3) is an important regulator of cAMP-mediated responses within the cardiovascular system. PDE3 exists as two subtypes: PDE3A and PDE3B, with distinct cellular and subcellular locations. Due to the lack of subtype-specific pharmacological tools, the definitive role of each subtype in regulating cardiovascular function has not been determined. In this study, we investigated platelet and cardiac function, using PDE3A and PDE3B gene knockout (KO) mice. Platelet-rich-plasma was prepared from the blood of KO and age-matched wild-type (WT) mice. PGE1 (1 microg/mL) almost completely inhibited aggregation of platelets from WT, PDE3A KO and PDE3B KO mice. In platelets from WT mice, cilostamide (100 microM), a selective PDE3 inhibitor, blocked collagen- and ADP-induced aggregation. In contrast, cilostamide had no effect on aggregation of platelets from PDE3A KO mice. In PDE3B KO mice, inhibition of collagen- and ADP-induced platelet aggregation was similar to that in WT mice. The resting intra-platelet cAMP concentration in platelets from PDE3A KO mice was twice that in the WT platelets. After PGE1 (0.1 microg/mL) stimulation, intra-cellular cAMP concentration was increased significantly more in platelets from PDE3A KO mice compared to WT mice. In vivo, PDE3A KO mice were protected against collagen/epinephrine-induced pulmonary thrombosis and death, while no such protection was observed in PDE3B KO mice. The heart rate of PDE3A KO mice was significantly higher, compared with age-matched WT mice, while that of PDE3B KO mice was similar to WT. There was no difference in cardiac contractility between PDE3A or PDE3B KO mice. Heart rate and contractility were increased in a similar dose-dependent fashion by isoproterenol in both types of KO mice. Cilostamide increased heart rate and contractility in WT and PDE3B KO but not in PDE3A KO mice. Compared to WT and PDE3B KO mice, cyclic AMP-PDE activity in membrane fractions prepared from the hearts of PDE3A KO mice was lower and not inhibited by cilostamide. The data suggest that PDE3A is the main subtype of PDE3 expressed in platelets and cardiac ventricular myocytes, and is responsible for the functional changes caused by PDE3 inhibition.[unreadable] [unreadable] In adipocytes, Phosphodiesterase 3B (PDE3B) is an important regulator of energy metabolism, including pathways regulated by insulin and cAMP-increasing hormones. Fractionation of membranes from 3T3-L1 adipocytes and primary murine adipocytes revealed that PDE3B was associated with plasma membrane caveolae(PM) and endoplasmic reticulum (ER)/Golgi fractions. Stimulation of 3T3-L1 adipocytes with insulin and the Beta3-receptor agonist CL316243 (CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. Insulin increased tyrosine phosphorylaiton of insulin receptor substrate-1 (IRS-1), and activated IRS-1-associated phosphatidylinositol 3-kinase (P13-K) and protein kinase B (PKB) in both intracellular membrane and cytosolic fractions. Superose 6 gel filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contain 32P-phosphorylated PDE3B and signaling proteins thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signaling proteins (14-3-3, PP2A, caveolin-1). CL-activated complexes contain Beta3-receptor, PKA-regulatory subunit (PKA-RII), and HSL. Insulin-induced macromolecular complexes apparently contain IRS-1, P13-K p85, PKB, HSP-90, 14-2-2. Confocal microscopy also indicated colocalization of PDE3B and PKB. Little or no insulin-receptor or PKA-RII co-eluted with the insulin-induced macromlecular complex(es); little or no IRS, PI3K or PKB co-eluted with CL-induced macromolecular complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, and insulin-induced phosphorylation/activation of PKB and PDE3B, as well as their co-immunoprecipitaiton and interaction during Superose 6 chromatography. Recombinant mouse (M)PDE3B co-immunoprecipitated and co-eluted during Superose 12 chromatography to a greater extent with recombinant p-PKB (phosphorylated/activated PKB) than dephospho PKB or p-deltaPKB (p-PKB lacking its PH domain). Co-immunoprecipitation of truncated recombinant MPDE3B proteins and p-PKB suggested that structural determinants for their interaction seem to reside in, or be regulated by, the N-terminal portion of MPDE3B.[unreadable] [unreadable] Phosphodiestease 3B, an important component of insulin and cAMP-dependent signalling pathways, has previously been shown to be activated by insulin and cAMP increasing hormones in adipocytes and hepatocytes. In order to study phosphorylation of PDE3B, we, in collaborative studies, used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes and H4IIE hepatoma cells. Phosphorylation of PDE3B after treatment of cells with insulin, cAMP-increasing agents, or the phosphatase inhibitor, calyculin A was analyzed by two-dimensional tryptic phosphopeptide mapping and mass spectrometry. We found that PDE3B is multisite phosphorylated in adipocytes and H4IIE hepatoma cells in response to all these stimuli. Several sites were identified; serine (S)273, S296, S421, S424/5, S474 and S536 were phosphorylated in adipocyte as well as H4IIE hepatoma cells whereas S277 and S507 were phosphorylated in hepatoma cells only. Several of the sites were phosphorylated by insulin as well as cAMP-increasing hormones indicating integration of the two signalling pathways upstream of PDE3B, maybe at the level of protein kinase B.[unreadable] [unreadable] Although mechanisms for acute activation of PDE3B and its potential physiological role(s) have been extensively studied in cultured cells, its role(s) in human and animal physiology or in the development of dysregulated metabolic states, including systemic insulin resistance, is not well understood. To evaluate the physiological role(s) of PDE3B, we induced a targeted disruption in the murine Pde3b gene by homologous recombination. Pde3b-KO mice exhibited multiple alterations in regulation of lipolysis, lipogenesis, and insulin secretion as well as signs of peripheral insulin resistance. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated lipogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide 1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The Beta3-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance.